2. 3D-printed titanium: a surface optimization analysis

Abstract

BACKGROUND CONTEXT Titanium was first used as an orthopaedic implant material in the 1950s and has been widely used in the decades following. Due to recent advancements in 3D-printing technology, titanium implants are now able to be manufactured with macroscopic, microscopic, and even nanoscopic precision. With this precision, surface optimization analysis can better elucidate the ideal implant interface for osseointegration. PURPOSE The purpose of this experiment is to examine the biological response of mesenchymal stem cells in three dimensions (3D) to titanium implants with different surface characterizations. STUDY DESIGN/SETTING Cell culture analysis. OUTCOME MEASURES Our outcome measures included cellular adhesion via environmental scanning electron microscope (ESEM) imaging, cell viability, and their biological response as determined by quantification of alkaline phosphatase, DNA, osteocalcin, BMP2, osteoprotegerin, and vascular endothelial growth factor (VEGF). METHODS 3D titanium scaffolds (7.5 mm x 10 mm x 7.5 mm) were printed with five variable approximate porosities of 40%, 50%, 60%, 70%, and 80%. Human bone marrow-derived mesenchymal stem cells (hMSCs) (ATCC, Manassas, VA) were seeded with an approximate initial density of 30,000 cells/cm2, incubated at 37°C, and fixed with 10% formalin. For early adhesion analysis, two scaffolds per porosity were fixed following a 72-hour culture and subsequently imaged by ESEM (Carl Zeiss Microscopy, Thornwood, NY, USA). One scaffold was imaged intact for surface analysis while the second scaffold was imaged following a single midline longitudinal cut to analyze internal cellular migration patterns. Cell viability determined by LIVE/DEAD assay was performed on a separate scaffold. The biological response was investigated on two separate scaffolds per group by cell lysate and media assays for alkaline phosphatase, DNA content, osteocalcin, BMP2, osteoprotegerin, and vascular endothelial growth factor (VEGF) quantification per manufacturer's instructions. RESULTS ESEM imaging analysis of each scaffold surface demonstrated that hMSCs had the strongest adhesive affinity for surfaces with porosities between 50% and 70%. Scaffolds within this range, when sliced longitudinally, demonstrated a more robust and dense internal cellular migration pattern than those outside the range. High cell viability on implant surface confirmed biological response by LIVE/DEAD assay. Conditioned media assay revealed increased levels of BMP2 expression on porosities between 50-70% with increased levels of VEGF, osteocalcin, and osteoprotegerin expression on scaffolds with porosities between 70-80%. Lower porosities scaffolds demonstrated increased levels of DNA and alkaline phosphatase activity. CONCLUSIONS Osseointegration requires complex biological conditions and our findings suggest that cellular optimization occurs with an approximate porosity of 60% (range: 50-70%). Made possible by the advancing field of 3D printing technology, precision surface engineering plays a determinant role in cellular adhesion and functioning, leading to enhanced osseointegrative potential. FDA DEVICE/DRUG STATUS Unavailable from authors at time of publication.